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Real-Time PCR Papers

An Introduction to Real-Time PCR
N.A. Saunders
The development of instruments that allowed real-time monitoring of fluorescence within PCR reaction vessels was a very significant advance. The technology is very flexible and many alternative instruments and fluorescent probe systems have been developed and are currently available. Real-time PCR assays can be completed very rapidly since no manipulations are required post-amplification. Identification of the amplification products by probe detection in real-time is highly accurate compared with size analysis on gels.
Real-Time PCR Platforms
M.J. Logan and K.J. Edwards
Real-time PCR continues to have a major impact across many disciplines of the biological sciences and this has been a driver to develop and improve existing instruments. From the first two commercial platforms introduced in the mid 1990s, there is now a choice in excess of a dozen instruments, which continues to increase. Advances include faster thermocycling times, higher throughput, flexibility, expanded optical systems, increased multiplexing and more user-friendly software.
Homogeneous Fluorescent Chemistries for Real-Time PCR
M.A. Lee, D.J. Squirrell, D.L. Leslie, and T. Brown
The development of fluorescent methods for a closed tube polymerase chain reaction has greatly simplified the process of quantification. Current approaches use fluorescent probes that interact with the amplification products during the PCR to allow kinetic measurements of product accumulation. These probe methods include generic approaches to DNA quantification such as fluorescent DNA binding dyes.
Performing Real-Time PCR
K.J. Edwards
Optimisation of the reagents used to perform PCR is critical for reliable and reproducible results. As with any PCR initial time spent on optimisation of a real-time assay will be beneficial in the long run. Specificity, sensitivity, efficiency and reproducibility are the important criteria to consider when optimising an assay and these can be altered by changes in the primer concentration, probe concentration, cycling conditions and buffer composition. An optimised real-time PCR assay will display no test-to-test variation in the crossing threshold or crossing point and only minimal variation in the amount of fluorescence.
Internal and External Controls for Reagent Validation
M.A. Lee, D.L. Leslie and D.J. Squirrell
PCR applications that require a high confidence in the result should be designed to control for the occurrence of false negatives. False negatives can occur from inhibition of one or more of the reaction components by a range of factors. While an external, or batch control is often used, the ideal control is one that is included in the reaction cocktail in a multiplex format. Early approaches used different sized amplicons combined with end-point analysis. Fluorescent homogenous real-time PCR methods have a number of advantages for implementing internal controls.
Quantitative Real-Time PCR
N.A. Saunders
Unlike classical end-point analysis PCR, real-time PCR provides the data required for quantification of the target nucleic acid. The results can be expressed in absolute terms by reference to external quantified standards or in relative terms compared to another target sequence present within the sample. Absolute quantification requires that the efficiency of the amplification reaction is the same in all samples and in the external quantified standards. Consequently, it is important that the efficiency of the PCR does not vary greatly due to minor differences between samples. Careful optimisation of the PCR conditions is therefore required. The use of probes in quantitative real-time PCR improves its performance and a range of suitable systems is now available.
Analysis of mRNA Expression by Real-Time PCR
S.A. Bustin and T. Nolan
The last few years have seen the transformation of the fluorescence-based real-time reverse transcription polymerase chain reaction (RT-PCR) from an experimental tool into a mainstream scientific technology. Assays are simple to perform, capable of high throughput, and combine high sensitivity with exquisite specificity. The technology is evolving rapidly with the introduction of new enzymes, chemistries and instrumentation and has become the "Gold Standard" for a huge range of applications in basic research, molecular medicine, and biotechnology.
Mutation Detection by Real-Time PCR
K.J. Edwards and J.M.J Logan
Real-time PCR is ideally suited for analysis of single nucleotide polymorphisms (SNPs) and has been increasingly used for this purpose since the advent of real-time PCR and as whole genome sequences have become available. It requires methods that are rapid, sensitive, specific and inexpensive, and several real-time methods have evolved which fulfil these requirements.
The Quantitative Amplification Refractory Mutation System
P. Punia and N.A. Saunders
The amplification refractory mutation system (ARMS), which has also been described as allele-specific PCR (ASP) and PCR amplification of specific alleles (PASA), is a PCR-based method of detecting single base mutations. ARMS has been applied successfully to the analysis of a wide range of polymorphisms, germ-line mutations and somatic mutations. The technique has the ability to discriminate low-levels of the mutant sequence in a high background of wild-type DNA. In an ARMS PCR the terminal 3' nucleotide of one of the PCR primers coincides with the target mutation. Most applications of the method rely on 'end-point' analysis, utilising the classic gel-electrophoresis method.
Real-Time NASBA
S. Hibbitts and J.D. Fox
NASBA is an isothermal nucleic acid amplification method that is particularly suited to detection and quantification of genomic, ribosomal or messenger RNA. The product of NASBA is single-stranded RNA of opposite sense to the original target. The first developed NASBA methods relied on liquid or gel-based probe-hybridisation for post-amplification detection of products. More recently, real-time procedures incorporating amplification and detection in a single step have been reported and applied to a wide range of targets. Thus real-time NASBA has proved to be the basis of sensitive and specific assays for detection, quantification and analysis of RNA (and in one case DNA) targets.
Applications of Real-Time PCR in Clinical Microbiology
A.D. Sails
The introduction of real-time PCR assays to the clinical microbiology laboratory has led to significant improvements in the diagnosis of infectious disease. There has been an explosion of interest in this technique since its introduction and several hundred reports have been published describing applications in clinical bacteriology, parasitology and virology. There are few areas of clinical microbiology which remain unaffected by this new method. It has been particularly useful to detect slow growing or difficult to grow infectious agents. However, its greatest impact is probably its use for the quantitation of target organisms in samples.
Application of Real-Time PCR to the Diagnosis of Invasive Fungal Infection
N. Isik and N.A. Saunders
The management of invasive fungal infections has been hampered by the inability to make a diagnosis at an early stage of the disease. Molecular diagnosis by PCR appears very promising since fungal DNA can be detected in the blood of infected patients earlier than when using conventional methods. Recently, interest in the diagnosis of invasive fungal infections by real-time PCR has increased. Real-time methods also have quantitative properties and are useful both for initial diagnosis and to assess the response to treatment. Many recent studies have combined serological tests with measurement of fungal DNA by using real-time PCR. Real-time PCR helps early diagnosis and arrangement of treatment protocols for patients with high risk of fungal infection.

General PCR Papers

Endonuclease-Mediated Long PCR and Its Application to Restriction Mapping
Curr. Issues Mol. Biol. (1999) 1: 77-88 Chengtao Her and Richard M. Weinshilboum
The polymerase chain reaction (PCR) is the most widely used technique for the study of DNA. Applications for PCR have been extended significantly by the development of "long" PCR.
A PCR-based Method for Isolation of Genomic DNA Flanking a Known DNA Sequence
Curr. Issues Mol. Biol. (1999) 1: 47-52 Catherine A. Boulter and Dipa Natarajan
A simple PCR-based method for the isolation of genomic DNA that lies adjacent to a known DNA sequence.
Universal TA Cloning
Curr. Issues Mol. Biol. (2000) 2: 1-7 Ming-Yi Zhou and Celso E. Gomez-Sanchez
TA cloning is one of the simplest and most efficient methods for the cloning of PCR products.
Analysis of Specific Bacteria from Environmental Samples using a Quantitative Polymerase Chain Reaction
Curr. Issues Mol. Biol. (2002) 4: 13-18 Clifford F. Brunk, Jinliang Li and Erik Avaniss-Aghajani
The use of quantitative PCR for measuring bacterial abundance in environmental samples.
PCR Clamping
Curr. Issues Mol. Biol. (2000) 2: 27-30 Henrik Ørum
An efficient, PCR based method for the selective amplification of DNA target sequences that differ by a single base pair. The method utilises the high affinity and specificity of PNA for their complementary nucleic acids and that PNA cannot function as primers for DNA polymerases.
DNA Splicing by Directed Ligation (SDL)
Curr. Issues Mol. Biol. (1999) 1: 21-30 DNA Yuri A. Berlin
Splicing by directed ligation (SDL) is a method of in-phase joining of PCR-generated DNA fragments that is based on a pre-designed combination of class IIS restriction endonuclease recognition and cleavage sites.